Mucosal surfaces throughout the body allow access to the external environment and present the opportunity for infection. A key part of the innate immune system is to prevent pathogen infection while limiting damage due to inflammation and oxidative stress. The paradox presented is how can one generate a reactive species that will damage a broad range of pathogens without harming self. To fulfill this paradox, a new paradigm on how the innate immune system uses thiocyanate (−SCN) as a precursor to generate hypothiocyanite (HOSCN), a broad-spectrum biocide that can be selectively metabolized by the host, is disclosed herein. The pseudohalides such as thiocyanate (−SCN) or selenocyanate (−SeCN) can directly react with neutrophil generated hypohalous acid such as hypochlorite (HOCl) to form hypothiocyanite/hyposelenocyanite (HOSCN/HOSeCN) (Thomas, E. L., and Fishman, M. (1986) Oxidation of chloride and thiocyanate by isolated leukocytes. J Biol Chem 261, 9694-9702) or can substitute for a halide like chloride as a preferred substrate for haloperoxidases such as myeloperoxidase (MPO) to form HOSCN/HOSeCN (van Dalen, C. J., et al. (1997) Thiocyanate and chloride as competing substrates for myeloperoxidase. Biochem J 327 (Pt 2), 487-492). Evolutionary divergence found in mammalian thioredoxin reductase (TrxR) supports the selective detoxification of HOSCN/HOSeCN through the incorporation of selenocysteine containing thioredoxin reductase (Sec-TrxR) that resists oxidative inactivation seen with cysteine containing thioredoxin reductases (Cys-TrxR) found in pathogenic bacteria (Hirt, R. P., et al. (2002) The diversity and evolution of thioredoxin reductase: new perspectives. Trends Parasitol 18, 302-308).
−SCN is obtained from the diet (Chandler, J. D., and Day, B. J. (2012) Thiocyanate: a potentially useful therapeutic agent with host defense and antioxidant properties. Biochem Pharmacol 84, 1381-1387) or formed as part of the detoxification of cyanide by sulfur transferases such as rhodanese (Maduh, E. U., and Baskin, S. I. (1994) Protein kinase C modulation of rhodanese-catalyzed conversion of cyanide to thiocyanate. Res Commun Mol Pathol Pharmacol 86, 155-173). −SCN can directly react with HOCl forming HOSCN (Thomas, E. L., and Fishman, M. (1986) Oxidation of chloride and thiocyanate by isolated leukocytes. J Biol Chem 261, 9694-9702). Both myeloperoxidase (MPO) and lactoperoxidase (LPO) can utilize −SCN to form HOSCN (van Dalen, C. J., et al. (1997) Thiocyanate and chloride as competing substrates for myeloperoxidase. Biochem J 327 (Pt 2), 487-492). The inventors have also seen that such reactions also occur with −SeCN that generate the antimicrobial agent hyposelenocyanite (HOSeCN).
Cystic fibrosis is a genetic disorder due to mutation and loss of function associated with the cystic fibrosis transmembrane conductance regulator (CFTR) protein (Riordan J R, et al. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073.). Cystic fibrosis is characterized by persistent lung infection with common bacteria including Pseudomonas aeruginosa (PA), Burkholderia cepacia complex (BCC), and methicillin-resistant Staphylococcus aureus (MRSA) (Lyczak J B, et al. 2002. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15:194-222). These pathogens frequently develop broad antibiotic resistance over the course of clinical care. The direct role for the mutant CFTR protein in the impairment of lung innate immunity is not known. CFTR protein regulates anion transport across apical epithelial cells (Quinton P M. 1983. Chloride impermeability in cystic fibrosis. Nature 301:421-422; Fragoso M A, et al. 2004. Transcellular thiocyanate transport by human airway epithelia. J Physiol 561:183-194; Tang L, et al. 2009. Mechanism of direct bicarbonate transport by the CFTR anion channel. J Cyst Fibros 8:115-121; Linsdell P, Hanrahan J W. 1998. Glutathione permeability of CFTR. Am J Physiol 275:C323-326). Among the endogenous anions that passivate through the CFTR channel are halides and pseudohalide such as chloride and thiocyanate (−SCN).
−SCN correlates with better lung function in patients with cystic fibrosis (Lorentzen D, et al 2011. Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions. Free Radic Biol Med 50:1144-1150). −SCN is concentrated up to mM amounts in secretions (e.g. saliva (Schultz C P, et al. 1996. Thiocyanate levels in human saliva: quantitation by Fourier transform infrared spectroscopy. Anal Biochem 240:7-12), nasal (Lorentzen D, et al 2011) and bronchoalveolar lining fluid (Gerson C, et al. 2000. The lactoperoxidase system functions in bacterial clearance of airways. Am J Respir Cell Mol Biol 22:665-671)). The CFTR protein is an important transporter of −SCN, which has been demonstrated to be decreased in apical secretions from CF airway epithelial cells (Fragoso M A, et al. 2004. Transcellular thiocyanate transport by human airway epithelia. J Physiol 561:183-194), CF animal model secretions (Gould N S, et al. 2010. Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury. Respir Res 11:119), and human CF saliva (Minarowski L, et al. 2008. Thiocyanate concentration in saliva of cystic fibrosis patients. Folia Histochem Cytobiol 46:245-246). −SCN is oxidized to HOSCN by haloperoxidases (e.g., myeloperoxidase (MPO), lactoperoxidase (LPO)) (Klebanoff S J, et al. 1966. The peroxidase-thiocyanate-hydrogen peroxide antimicrobial system. Biochim Biophys Acta 117:63-72). HOSCN is a selective oxidant preferring low pKa thiols and selenols (RSH and RSeH) (Skaff O, et al. 2012. Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage. Biochem J 441:305-316) and is used by the immune system to kill bacteria (Thomas E L, et al. 1994. Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci. Infect Immun 62:529-535), fungus (Majerus P M, Courtois P A. 1992. Susceptibility of Candida albicans to peroxidase-catalyzed oxidation products of thiocyanate, iodide and bromide. J Biol Buccale 20:241-245) and viruses (Mikola H, et al. 1995. Inhibition of herpes simplex virus type 1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated hypothiocyanite. Antiviral Res 26:161-171; Pourtois M, et al. 1990. Inhibition of HIV infectivity by lactoperoxidase-produced hypothiocyanite. J Biol Buccale 18:251-253). The inventors have previously shown that mammalian selenocysteine-containing TrxR (Sec-TrxR), which contains a selenocysteine, can metabolize HOSCN in human lung epithelia (Chandler J D, et al. 2013. Selective metabolism of hypothiocyanous acid by mammalian thioredoxin reductase promotes lung innate immunity and antioxidant defense. J Biol Chem 288:18421-18428). The bacterial form of TrxR lacks selenocysteine and cannot metabolize HOSCN and instead is inactivated by HOSCN (Chandler J D, et al. 2013. Selective metabolism of hypothiocyanous acid by mammalian thioredoxin reductase promotes lung innate immunity and antioxidant defense. J Biol Chem 288:18421-18428).
Selenocyanate (−SeCN) is a chemical analog of endogenous −SCN. Substitution of selenium for sulfur in a compound usually results in a more reactive compound (Caldwell K A, Tappel A L. 1964. Reactions of Seleno- and Sulfoamino Acids with Hydroperoxides. Biochemistry 3:1643-1647). The inventors believe that −SeCN is treated by the body in a similar manner as −SCN and will be converted by haloperoxidases to a more potent antimicrobial oxidant, hyposelenocyanite (HOSeCN) while keeping its ability to be selectively detoxified by the host selenocysteine containing TrxR.
There is significant mortality and morbidity from pulmonary infections in many chronic lung disease most notability occurring in CF. The hallmark of CF is a dysregulation of lung inflammation and compromised ability to resolve infection and inflammation (Cantin, A. (1995) Cystic fibrosis lung inflammation: early, sustained, and severe. Am J Respir Crit Care Med 151, 939-941). As disclosed herein the inventors demonstrate that impaired transport and utilization of −SCN disrupts the ratio of hypothiocyanite (HOSCN)/hypochlorite (HOCl) and sets up for the spiraling cycle of infection, chronic inflammation, and oxidative stress that are the major drivers of CF lung disease. As disclosed herein, the characterization of a novel selective detoxification pathway in lung host defense is undertaken. In addition, a better understanding of the importance and linkage between host defense and excessive lung inflammation is determined; the importance of −SCN/HOSCN in pulmonary bacterial host defense and airway inflammation is assessed; and development of a novel therapeutic selenocyanate (−SeCN) to treat infectious lung disease by promoting antimicrobial defense and suppressing inflammation is shown.